We've developed a new technology based on micro-molded nonadhesive agarose to guide the self assembly of 3D micro-tissues. Mono-disperse cells are pipetted into the micro-mold, they settle into the molded features, are unable to bind to the nonadhesive agarose and so spontaneously self assembly 3D micro-tissues. By making complex features in the micro-molds, we've shown for the first time that cells will self assembly complex structures such as rods, toroids and honeycombs. Little is understood about the rules that govern this directed cellular self-assembly process, especially for complex branching structures and so the overarching goal of this proposal is to define and quantify these rules. Our hypothesis is that the rules of selfassembly differ between specific cell types and that in addition to surface adhesive forces, cytoskeletal mediated tension/contraction contribute to cell specific differences and significantly influence the directed self assembly of complex micro-tissues. To test this hypothesis and define the rules of directed self assembly, we propose to use five cell lines and their mixtures, normal human fibroblasts (NHF), a hepatocyte cell line (H35), smooth muscle cells (SMC) and two endothelial cell lines;human umbilical cord vein endothelial cells (HUVEC) and calf pulmonary artery endothelial cells (CPAE) to test the following specific aims: Aim 1. To determine cell type differences in directed self assembly and their ability to generate complex shapes. Aim 2. To quantify the respective roles of cytoskeletal mediated contraction and cell surface adhesion proteins on directed self-assembly. Modified Public Health Relevance Using micro-molded agarose, we have developed a novel and surprisingly easy system for creating 3D micro-tissues with complex shapes such as rods, toroids and honeycombs that are scaffold-free. 3D micro-tissues made in this way may have applications in regenerative medicine, tissue engineering and drug development. Modified Specific Aims Three dimensional (3D) tissue model systems have applications in regenerative medicine, tissue engineering and drug development and we have developed a novel and surprisingly easy system for creating 3D tissues with complex shapes that are scaffold-free. Mono-dispersed cells are seeded onto nonadhesive micro-molded agarose where the cells settle and then aggregate into 3D micro-tissues. The geometry of the micromold can direct the self-assembly of a complex cellular structure such as toroids and honeycomb structures. Little is understood about the rules that govern this directed cellular self-assembly process, especially for complex branching structures and so the overarching goal of this proposal is to define and quantify these rules. Such rules will form the foundation for building ever more complex microtissues for use in numerous applications including tissue engineering and cell biology. Our hypothesis is that the rules of self-assembly differ between specific cell types and that in addition to surface adhesive forces, cytoskeletal mediated tension/contraction contribute to cell specific differences and significantly influence the directed selfassembly of complex microtissues. To test this hypothesis and define the rules of directed self-assembly, we propose the following specific aims: Aim 1. To determine cell type differences in directed self-assembly and their ability to generate complex shapes. Using micromolded agarose, we've shown for the first time that mono-dispersed cells can selfassemble microtissues more complex than the spheroid, a geometry that minimizes the surface area to volume ratio, that maximizes intercellular adhesion and minimizes surface energy. In fact, cells have the ability to selfassemble into microtissues with complex geometries (rods, toroids and lumen-containing honeycombs) and that this ability varies with cell type. Five cell lines will be tested, normal human fibroblasts (NHF), a hepatocyte cell line (H35), human smooth muscle cells (SMC) and two endothelial cell lines;human umbilical cord vein endothelial cells (HUVEC) and calf pulmonary artery endothelial cells (CPAE). The properties of directed selfassembly will be determined by measuring the ability of cells to form several model shapes of increasing complexity (spheroid, rod, toroid, loop ended dog bone and honeycomb). Each of these shapes enables us to quantitatively measure fundamental properties of directed self-assembly and measure cell differences. Beyond the spheroid, (the shape that minimizes energy and surface area), can cells self-assemble into a rod and what is the aspect ratio (length/width) of the steady state structure? Can cells form a stable toroid structure, (a rod wrapped around a central hydrogel peg) and what are the differences between the unconstrained and constrained structures, (rod vs toroid)? Can cells form a loop ended dog bone structure (2 toroids connected by a rod) and how do changes to the geometry of the toroids and connecting rod influence this structure? Can cells selfassemble a radial honeycomb, a complex branching structure with patent lumens (Fig 1) and if not, (as our preliminary data for NHFs shows) where in the radial structure does it fail and how is this related to points of stress? Since natural tissues are mixtures of different cell types we will also test combinations of cells to determine how cells sort and cross-modulate the directed self-assembly process. Aim 2. To quantify the respective roles of cytoskeletal mediated contraction and cell surface adhesion proteins on directed self-assembly. New mathematical models are needed to understand the contributions of cell to cell adhesion as well as cytoskeletal mediated tension in self-assembly, especially with regards to microtissue shape and stability. Using a variation of a toroid micromold where the central peg is a conical pillar, we've shown that toroidal microtissues will move up the cone during self-assembly. This geometry lends itself to a mathematical model and when coupled to experimental data, our latest advance shows that we can measure J, the surface energy per unit area of the outer surface of the microtissue. Surface energy has never been measured in self assembly. We propose to measure J for different cell types and cell mixtures. To quantify the relative contributions of specific proteins to J, we will also perform systematic studies to inhibit (blocking MAb and drugs) cell surface adhesion molecules (i.e., cadherins and integrins) and cytoskeletal proteins (i.e., microtubulules, microfilaments, intermediate filaments, myosin II) and measure, for the first time, their respective contributions to self assembly. These data will provide a new mathematical model with important quantitative insight into self-assembly as well as new molecular based strategies to control microtissue design.